A significant barrier to successful non-viral gene therapy results from the dramatic decrease in foreign transgene expression after introduction in mammalian systems. Many questions have been asked about chromatin modulation in gene silencing, but there is little knowledge of what exactly controls where nucleosomes sit on DNAand how this knowledge can be applied to therapeutic vector design. Hemophilia B is an excellent candidate for successful gene therapy because this disease is caused by deficiency of a single gene, factor IX. The overall aim of this work is to develop non-integrating factor IX vectors with sustained expression through manipulation of the chromatin state. Putative nucleosome positioning and excluding signals will be added at strategic positions in factor IX expression vectors, and the constructs will then be delivered to the mouse liver by hydrodynamic tail vein injection. Vector design will be guided by information gained from nucleosome core occupancy maps and chromatin immunoprecipitation assays. We have developed a DNA hybridization technique, in combination with the Solexa high-throughput sequencing platform, to selectively map vector DNA nucleosome positions in vivo. This novel method is the first example of high-resolution mapping of global chromatin structure in a delivered vector. These aims would start to give a mechanistic answer to what happens when foreign DNA enters a cell. In the future, this work could improve Hemophilia B gene therapy applications by supplying broadly applicable guidelines for how to influence nucleosome occupancy to sustain expression of a delivered gene, regardless of the vector type. Relevance: The work proposed here seeks to improve gene therapy for Hemophilia B and other diseases where a necessary gene is missing. We aim to create better agents of DNA replacement that will correct the disease through infrequent administration of a DNA therapeutic.